Multi-functional micro and nanoparticles for use in root canal therapies

ABSTRACT

Chitosan nanoparticles are provided for use in the in vivo treatment of connective tissues in root canal therapies. The nanoparticles are optionally linked with one or more photoactivatable compounds for providing antibacterial/antibiofilm properties, neutralizing bacterial byproducts and/or chemical/photodynamic crosslinking to achieve enhanced mechanical properties, chemical stability in connective tissues and/or to improve surface/interfacial integrity between filling material and connective tissue.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/385,596, which is the national phase application claiming the benefitof PCT/CA2013/000275 filed 21 Mar. 2013, in English, which furtherclaims the benefit of 35 USC §119(e) to U.S. Provisional ApplicationSer. No. 61/614,235 filed 22 Mar. 2012.

SCOPE

The invention relates to nanoparticles for use in the in vivo treatmentof connective tissues in root canal therapies, and more particularlychitosan polymer nanoparticles which may optionally be linked with oneor more photoactivatable compounds for providingantibacterial/antibiofilm, neutralize bacterial byproducts and/orchemical/photodynamic crosslinking to achieve enhanced mechanicalproperties, chemical stability in connective tissues and/or chemicals toimprove surface/interfacial integrity between filling material andconnective tissue.

BACKGROUND

Root canal or endodontic therapies involve the physical removal of thetooth root pulp by using successively larger helical dental files andreamers. The files and reamers penetrate and remove the pulp tissueleaving a hollowed-out root canal or opening which is bordered byexposed dentin. The root canal preparation extends to the apical tip ofthe root to allow infection drainage and prevent re-infection. Inconventional root canal therapies following removal of the dental pulpand cleaning, a suitable packing material, such as gutta-percha rubber,is inserted into the hollowed-out root canal in conjunction with acement and/or sealer, and thereafter heat fused in place. After thegutta-percha filing, the tooth is covered with a crown, amalgam, orcomposite dental restoration filling material.

Because the infection and subsequent removal of the highly hydratedvascularized dental pulp tends to weaken the remaining dentin structure,teeth which undergo endodontic therapies may be more prone to fractureand root failure. In addition, the surrounding dentin itself isgenerally subject to infection infiltration from the infected pulp, viadentinal tubules.

In endodontic treatments, infected hard tissues within the root dentinare typically managed using antimicrobial agents which are selected toeliminate causative microorganisms. Conventional treatment approachesfrequently result in the incomplete elimination of microbes which residewithin the complexities of the root dentin. As well, conventionalantimicrobial agents may result in treatment induced changes in themechanical characteristics of the dentin tissue surrounding thehollowed-out root canal, as well as degradation of the infected dentintissues due to host and/or bacterial derived proteases. Degradation ofthe hard dentin tissues may further result in a significant decrease ofmechanical tooth strength. While various alternative antimicrobialapproaches to achieve effective root dentin disinfection have beenproposed, heretofore, conventional root therapies have not addressed theimprovement of mechanical properties of infected hard tissues.

Photodynamic therapy involving the exposure of tissues to selected lightenergies has been adopted for use in multiple treatment applications,including antibacterial disinfection, anticancer therapies, tissuewelding and tissue engineering. A combination of a light-activatablechemicals (photosensitizers), appropriate light energy (UV) and oxygenare often important factors in photodynamic therapy (PDT) basedtreatments. With photodynamic therapy, the photosensitizer is excited byillumination with appropriate wavelength and goes to a higher-energy‘triplet state’ from a lower-energy ‘ground state’. Most typically, theexcited photosensitizer molecules transfer electrons to neighboringmolecules (type-1 reaction) to generate radical oxygen species, or itsenergy to the ground state molecular oxygen (type-2 reaction) togenerate highly reactive oxygen species (ROS), and most typicallysinglet oxygen.

Photodynamic therapy may also be useful in biomedicine for thephotodynamic crosslinking of proteins and collagen. The singlet oxygenproduced facilitates formation of inter and intramolecular covalentcrosslinks in collagen molecules and other available active sites in thepresence of appropriate photosensitizers such as Rose Bengal (RB).Photodynamic crosslinking is a rapid process, resulting from thegeneration of reactive oxygen species and formation of covalent collagencrosslinks in a light-independent manner. Covalent coupling between freeamino groups and photo-oxidized amino acids have been proven by thedecrease in reactivity and available free amino groups followingphotodynamic crosslinking in the presence of a sensitizer. The formationof additional crosslinks resulted in improved biological and mechanicalproperties of collagen structures. Incorporation and crosslinking ofbiopolymers, such as elastin and chitosan (CS) with collagenadvantageously may reinforce the collagen scaffolds. Photosensitizersolutions are however, generally known to be susceptible to leavingresidual traces after photoactivation, which may not be acceptable inthe in vivo treatment of tissues or applications.

The uptake of anionic and cationic photosensitizers is known to occurvia different mechanisms. Anionic photosensitizers such as Rose Bengaladhere only superficially. Deeper penetration into bacterial cells orthrough the highly negative extracellular polysaccharide is not possibleand uptake may be increased in the presence of divalent cations.Conjugation of anionic photosensitizers with poly-Llysine and polymyxinB nonapeptide have been tried to increase the antibacterial efficacyagainst both gram-positive and gram-negative bacteria. Immobilization ofRose Bengal on polystyrene beads has also shown antibacterial propertieswhen irradiated. Although photosensitizers have been conjugated withdifferent readily available synthetic polymers and liposomes, these alsopossess a significant limiting factor of biocompatibility when appliedin-vivo.

SUMMARY

The applicant has appreciated that the immobilization ofphotosensitizers on polymeric supports could avoid or minimize theformation of residual photosensitizers, making such compounds moresuitable for use in in vivo. Further, immobilization of polymericsupports may also provide the added advantage of enhanced stability incase of physiologic environments. Although photosensitizers have beenconjugated with different readily available synthetic polymers andliposomes, these possess a significant limiting factor ofbiocompatibility when applied in vivo. The applicant has recognized thatthe use of naturally occurring biopolymers, such as chitosan may howevercounteract the biocompatibility issues.

Chitosan is a linear polysaccharide, a derivative of chitin, is anabundant natural biopolymer, and has received significant interest foruse in biomedicine, food industries, agriculture and environmentalfields. Chitosan shows a broad range of antimicrobial activity,biocompatible and biodegradable properties. The chitosan polymers withits large number of free hydroxyl and amino groups has been used forvarious chemical modifications and grafting. Chitosan polymers arewettable, favouring intimate contact between the sensitized surface orphotosensitizer and aqueous suspensions of microorganisms. Chitosanpolymers are also considered to be structurally similar to extracellularmatrix components and can be used to reinforce collagen constructs.

A disadvantage of chitosan is its low solubility at a physiological pHof 7.4 due to its rigid crystalline structure and primary amino groupresidues. However, the applicant has appreciated that conjugation ofchitosan with photosensitizers such as Rose Bengal, or other anionicallyor cationically charged photosensitizers may result in water-solubleparticles at even higher pH levels.

Accordingly, one objective of the present invention provides for anantibacterial composition for use in vivo in pre-treating hard and/orconnective tissues to minimize and/or reduce the possibility ofbacterial infection/reinfection therethrough. In addition, it is alsoknown that chitosan requires (more than 24 hours) to eliminatefree-floating bacteria and is not able to disrupt bacterial biofilms,which is important in the treatment of root canal treatment.

Another objective of the invention is to provide a composition forenhancing the fracture toughness and/or mechanical strength of hardand/or connective tissues in the body, and more preferably dentintissues.

A further object of the invention is to provide improved nanoparticleshaving a size selected at less than about 100 nanometers, and preferablyfrom about 60 nanometers to 90 nanometers, and which are suitable foruse in sealing and/or strengthening hard connective tissues in the body,and more preferably strengthening tooth dentin as part of restorativeendodontic or restorative treatments.

In one aspect, the present invention utilizes multifunctionalchitosan-based particles which preferably have micro/nano dimensions ofup to 150 nanometers, preferably less than about 100 microns and mostpreferably about 60 to about 90 nanometers, and which are preferablyadmixed with a pharmaceutically acceptable carrier. The particles may ormay not be photoactivatable, whereby exposure to light energy andpreferably a selected light energy (for example in green light) may beused to effect crosslinking and/or generate antibacterial radical oxygenspecies. In one preferred use, the composition is to be applied to aroot canal wall/dentin of a tooth following root exposure or anendodontic root procedure, and before filling of the sealer and rootcanal obturation material, such as gutta-percha rubber.

In another preferred use, the invention provides for the in vivoapplication to hard or connective tissues of a composition comprisingchitosan and/or chitosan-based derivatives in micronized particle form,and preferably a composition which comprises chitosan-based particles inan amount of 0.3 to 1% (by weight). More preferably, the chitosan basednanoparticles are conjugated/functionalized with a photoactivatablecarrier, such as Rose Bengal for use to:

-   (a) Inactivate and/or inhibit activation of residual microbes and    biofilms;-   (b) Inhibit hard tissue, and preferably dentin surface degradation    (resorption) by enhancing its chemical stability;-   (c) Inhibit microbial re-entry (bacterial adherence, bacterial    penetration via interfaces into hard and/or connective tissue);-   (d) Improve mechanical integrity of connective and/or hard tissue,    and preferably dentin (fracture toughness); and/or-   (e) Improve connective tissue or dentin-obturating material    interface by biomineralization.

More preferably, the invention provides bioreactive micro ornanoparticles for use in inhibiting one or more of the prevention ofbacterial persistence, bacterial reentry/recolonization, ultrastructuralchanges, degradation and/or compromise in the mechanical characteristicsof connective or hard tissues, and more preferably dentin inendodontically treated teeth.

In another aspect, the present invention provides multifunctionalbioactive micro nanoparticles for use in vivo in the enhancement of oneor more antibacterial properties, interfacial integrity and/or fracturetoughness of infected dentin hard tissues in root canal therapies.

Chemical or photodynamic crosslinking methods have been used in tissueengineering to stabilize collagenous biological tissues by inducingvarious intra and intermolecular crosslinks in collagen. The applicanthas appreciated dentin as a biocomposite, contributes to the structuralstability of the root treated teeth and can be stabilized by collagencrosslinking processes. Apart from the cytotoxicity of glutaraldehyde,the treatment time required to establish stable collagen crosslinkstends to be much longer with chemical crosslinking methods, and is amajor limitation especially for in vivo clinical applications, whereshorter treatment time is highly desirable and biocompatibility is ofconcern.

Photodynamic crosslinking advantageously may provide a rapid processthat occurs via the production of singlet oxygen or radicals by thelight excited photosensitizers. The singlet oxygen interacts withphotooxidizable amino acid residues, such as Cysteine, Histidine,Tryptophan or Tyrosine in a protein molecule. The photooxidizedproducts, in turn, react with normal or photoaltered residues in anotherprotein molecule resulting in a crosslink. The addition of polymers,such as chitosan, during collagen crosslinking may advantageously beused in in vivo methods to produce collagen scaffolds in tooth dentinwith superior biological and physical properties.

It has been appreciated that nanoparticles of photoactivated chitosanpolymer-Rose Bengal (CSRBnp) not only will induce crosslinking ofcollagen, but also allow covalent bonding of chitosan with the collagenmatrix. Accordingly, in another aspect of the invention, chitosan iscombined with photosensitizers such as Rose Bengal to aid in developingmultifunctional nanoparticles which possess enhanced antibacterialefficacy, induce crosslinking of collagen matrix, and/or facilitate itsincorporation into the collagen matrix of the dentin duringphotoactivation. In addition, the composition could be applied duringthe restorative treatments and/or the minimally invasive management ofdentinal caries.

Chitosan polymer-Rose Bengal nanoparticles provide both the broad rangeof antimicrobial properties of chitosan and photosensitizer propertiesof Rose Bengal. Further, nanosized particles having a size selected atbetween about 60 nm and 90 nm advantageously provide a reactive surfacearea which increases aiding to the antibacterial effect. Chitosanpolymer-Rose Bengal nanoparticles may thus perform dual function ofenhanced elimination of bacterial biofilm and improved structuralstability/mechanical reinforcement of dentin collagen followingphotodynamic crosslinking.

Most preferably, the multifunctional chitosan nanoparticles consist of aphotosensitizer which is conjugated with chitosan and/or phosphorylatedchitosan combined with photo-activation, and which are provided as acompound or in a composition for use in a single step root canaldisinfection procedure immediately before root canal filling andsealing.

Accordingly, in one aspect the present invention resides in acomposition for use in in vivo disinfection and/or remineralizationtreatment of connective or hard tissues comprising nanoparticles, saidnanoparticles comprising a polysaccharide having a plurality of five orsix membered ring carbohydrate monomers, where each said monomer isoptionally substituted with at least one of a primary amine and asecondary or tertiary amine having an acyl group with two to sevencarbon atoms.

In another aspect, the invention resides in a method of dentalrestoration whereby following exposure of dentin, contacting the dentinwith aforementioned composition.

In a further aspect, the present invention resides in a method of makinga medicament for in vivo disinfection and remineralization of hard orconnective tissues, comprising: forming a phosphorylated chitosanpolymer; and micronizing said phosphorylated chitosan polymer to formnanoparticles having an average size selected at between about 40microns and about 80 microns, and admixing said nanoparticles with apharmaceutically acceptable carrier.

In yet another aspect, the present invention resides in a method ofmaking a medicament for in vivo disinfection and remineralization ofhard or connective tissues, comprising: forming a phosphorylatedchitosan polymer; and micronizing said phosphorylated chitosan polymerto form nanoparticles having an average size selected at up to about 100nanometers, and preferably between about 60 nanometers and 90nanometers, and admixing or mixing said nanoparticles with apharmaceutically acceptable carrier.

The present disclosure provides a composition for endodontic or dentalrestorative use, comprising:

multifunctional biopolymer-based particles wherein a first portion ofbiopolymer-based particles have biopolymer repeat units covalentlyfunctionalized with photosensitizer moieties and a second portion of theof biopolymer-based particles have biopolymer repeat units covalentlyfunctionalized with phosphorylated moieties; and

a pharmaceutically acceptable liquid carrier, wherein themultifunctional biopolymer-based particles are mixed with thepharmaceutically acceptable liquid carrier to form a slurry.

The present disclosure provides a method of dental treatment,comprising:

contacting a dentin of a tooth with a pharmaceutical compositioncomprising a slurry of biopolymer-based particles wherein a firstportion of biopolymer-based particles have biopolymer repeat unitscovalently functionalized with photosensitizer moieties and a secondportion of the of biopolymer-based particles have biopolymer repeatunits covalently functionalized with phosphorylated moieties; and

exposing the slurry of biopolymer-based particles to a light having awavelength selected to activate the photosensitizer moieties for aperiod of time sufficient to crosslink the biopolymer-based particlesfunctionalized with the photosensitizer moieties to the dentin.

The present disclosure provides a method of dental treatment,comprising:

removing infected pulp tissue from a tooth root and forming ahollowed-out root canal thereby exposing dentin along a substantiallength of the hollowed-out root canal;

applying a slurry of biopolymer-based particles to the exposed dentinwithin the hollowed-out root canal, wherein a first portion ofbiopolymer-based particles have biopolymer repeat units covalentlyfunctionalized with photosensitizer moieties and a second portion ofbiopolymer-based particles have biopolymer repeat units covalentlyfunctionalized with phosphorylated moieties;

exposing the slurry of biopolymer-based particles to a light having awavelength selected to activate the photosensitizer moieties for aperiod of time; and

after exposing the slurry to the light, filling the hollowed-out rootcanal with a filling material.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference may now be had to the following detailed description takentogether with the accompanying drawings in which:

FIG. 1 shows the chemical reaction both during conjugation of chitosannanoparticles chitosan with Rose Bengal in the presence of EDC(N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide) and NHS(N-Hydroxysuccinimide), wherein the formation of chemical bonds betweenthe NH group of chitosan and photosensitizers are highlighted withdotted circles;

FIG. 2 shows schematically the mechanism of phosphorylation of chitosan;

FIG. 3 shows graphically FTIR analysis of phosphorylated chitosan (a)chitosan (b) phosphorylated chitosan;

FIG. 4 shows graphically ATR-FTIR analysis of dentin surface: (a), thecontrol sample; (b) specimen coated with phosphorylated chitosan, (c)specimen crosslinked with phosphorylated chitosan (d) specimen coatedwith phosphorylated chitosan after remineralization usingnon-fluoridated remineralizing solution; (e) untreated specimen afterremineralization using fluoridated remineralizing solution; (f) specimencrosslinked with phosphorylated chitosan after remineralization usingnon-fluoridated solution; (g) sound dentin specimen with smear layer;

FIG. 5 shows graphically XRD diffraction of the surface of dentinspecimen: (a), the control specimen; (b) specimen coated withphosphorylated chitosan after remineralization using non-fluoridatedremineralization solution; (c) untreated specimen after remineralizationusing fluoridated remineralization solution; (d) specimen cross-linkedwith phosphorylated chitosan after remineralization usingnon-fluoridated remineralizing solution, (e) sound dentin specimen withsmear layer;

FIG. 6 shows SEM results of remineralization of partially demineralizeddentin collagen (a) control sample; (b) sample coated withphosphorylated chitosan after remineralization using non-fluoridatedremineralization solution (e) sample cross-linked with P-chi afterremineralization using non-fluoridated remineralizing solution; (f)highly magnified image of (e) showing petal-like mineral crystals;

FIG. 7 shows confocal Laser Scanning Microscopy microscopic Imagesshowing significant biofilm proliferation at the sealer-dentin interfacein the control sample, and with no conspicuous biofilm proliferation atthe sealer-dentin interface, where the dentin were conditioned with thephosphorylated chitosan nanoparticles;

FIG. 8A shows a transmission electron microscopy image of chitosanpolymer-Rose Bengal nanoparticles with the inset showing enlarged view(scale bar=100 nm). The chitosan polymer-Rose Bengal nanoparticles wereof 60±20 nm in size;

FIG. 8B shows a typical graph showing absorption spectrum of Rose Bengaland chitosan polymer-Rose Bengal nanoparticles. The absorption peak at550 nm was not affected following conjugation of chitosan with RoseBengal. (C) FTIR spectra of chitosan and chitosan polymer-Rose Bengal(400-4000 cm⁻¹ wavenumber). The amide peaks and presence of saccharidepeak confirmed the conjugation of chitosan with Rose Bengal;

FIG. 9 shows graphically the oxidation of DPBF due to singlet oxygengeneration following photoactivation of Rose Bengal and chitosanpolymer-Rose Bengal nanoparticles measured as the reduction of DPBFabsorbance;

FIG. 10 shows a graph showing cell survival following treatment withRose Bengal and chitosan Rose Bengal nanoparticles with and withoutphotodynamic therapy. Photodynamic therapy resulted in significantlyincreased cytotoxicity as compared to chitosan Rose Bengal nanoparticlestreatment without photodynamic therapy. (p<0.05);

FIG. 11 illustrates a graph showing the release of cell constituents(absorbance at 260 nm) following treatment with Rose Bengal and chitosanRose Bengal nanoparticles with and without photodynamic therapy.Chitosan polymer-Rose Bengal nanoparticles at higher concentrationshowed inherent ability to induce bacterial membrane damage;

FIG. 12 shows graphically log number of E. faecalis in planktonic (A)and biofilm (B & C) forms surviving the photodynamic therapy conductedin a multiwell plate. There was a significant difference in the killingby chitosan polymer-Rose Bengal nanoparticles compared to Rose Bengal.Error bars show the standard deviation from average value;

FIG. 13 shows three-dimensional confocal laser scanning microscopyreconstruction of the E. faecalis biofilm subjected to photodynamictherapy using Rose Bengal and chitosan polymer-Rose Bengalnanoparticles. (Inlet shows the sagittal section) (60×). (A) The biofilmreceiving no treatment, (B) the biofilm subjected to sensitization withRose Bengal, (C) the biofilm subjected to sensitization with Rose Bengalfollowed by irradiation (40 J/cm²), (E) the biofilm subjected tosensitization with chitosan polymer-Rose Bengal nanoparticles, and (F)the biofilm subjected to chitosan polymer-Rose Bengal nanoparticles andirradiation (40 J/cm²);

FIG. 14 shows schematically FTIR spectra of dentin-collagen (A);enzymatic degradation of dentin-collagen (B); and stress-strain curveafter mechanical testing of dentin-collagen following crosslinking (C);

FIG. 15 shows transmission electron micrographs of dentin collagenwithout any treatment (A & B) and following photo-crosslinking treatmentwith chitosan polymer-Rose Bengal nanoparticles (C & D);

FIG. 16 shows graphically the log number of E. faecalis in biofilm formssurviving the photodynamic therapy (PDT) conducted in a multiwell plate.FIG. 16 shows there was a significant difference in antibacterialproperties achieved by chitosan Rose Bengal nanoparticles as compared toRose Bengal. The error bars illustrate the standard deviation for theaverage value;

FIG. 17 shows the log number of E. faecalis in planktonic formssurviving different antibacterial treatments in the presence of tissuesinhibitors. Chitosan nanoparticle effect was inhibited significantly bybovine serum albumin (BSA) 2% even following 24 hour treatment.

FIG. 18 shows chitosan polymer rose Bengal nanoparticles afterphotodynamic therapy followed by a longer period of interactionresulting in complete elimination of E. faecalis even in the presence ofbovine serum albumin. Arrow bars show the standard deviation fromaverage values; and

FIG. 19 shows AFM images of planktonic E. faecalis (a) control, withoutany treatment; (b) chitosan treated cells for 15 minutes; and (c)Chitosan polymer Rose Bengal nanoparticles treated cells for 15 minutesfollowed by photodynamic therapy (5 J/CM²). The controlled cells showedsmooth celled surface with definite cell membrane. Chitosan nanoparticletreated cells were covered by the nanoparticles and the cell surfaceappeared rough. Following treatment with Chitosan polymer Rose Bengalnanoparticles and photodynamic therapy, the cell surface appearedcorrugated, nanoparticles were seen bound and penetrating theantibacterial cells. The cell membrane was defuse and regular,suggesting membrane damage.

DETAILED DESCRIPTION

The present invention provides for a modified polymeric photosensitizercomposition that includes photoactivatable nanoparticles of a chitosanpolymer which have been conjugated with Rose Bengal as aphotosensitizer. Preferably, the nanoparticles have a size selected atup to about 100 nanometers, preferably between about 60 nanometers and90 nanometers, and more preferably, about 80 nanometers. The particlesare preferably admixed with a suitable liquid carrier to form a slurryand which, as will be described, act as an antibacterial andremineralization agent for pre-treating the dentin of hollowed-out rootcanal, prior to placement and cementation of gutta-percha fillermaterial within the hollowed-out tooth root canal in an endodontictherapy. The carrier may be any one of water and an alcohol.

In an endodontic treatment using the nanoparticles of the presentinvention, the dental pulp canal is first exposed. The pulp of theinfected root is removed using endodontic files in a conventional mannerand allowed to drain. Following the pulp removal, the infected materialfrom the root canal is removed and root canal space is shaped usingmechanical instruments and chemical irrigants. Once this cleaning andshaping procedure of the root canal system is completed, thenanoparticles slurry (supplied in a packet) can be applied within theroot canal space by syringe with or without agitation/activation usingultrasonic/sonic methods. The chitosan conjugated photosensitizernanoparticles will be activated using light. A fiber optic cable will beused to deliver light into the root canal is blot-dried using paperpoints. Following blot-drying the root canal is exposed to light energyhaving a wavelength selected to photoactivate the nanoparticles for asufficient period of time to achieve the desired cross linking and/orantibacterial effect. Most preferably, the slurry is exposed to visiblelight energy for a period of up to 10 minutes and more preferably fromabout 3 to 8 minutes. After cross-linking, the root canal is filledusing conventional root canal obturating/filling material and root canalsealer.

In particular, following the activation of the chitosan polymer RoseBengal nanoparticles, the root canal is washed.

A gutta-percha cone is coated in a cement mixture of zinc oxide eugenolbased cements, methacrylate based cements or epoxy based cements.Thereafter, the coated gutta-percha cone is physically placed within thehollowed-out, cleaned and shaped root. After placement, the case isthermally fused in place using a heated packing tool.

Following root filling with gutta-percha, the exposed dentin pulpchamber is covered with an amalgam or composite filling material, andprosthetic crown thereafter is applied.

In accordance with the preferred application, chitosan polymer-RoseBengal nanoparticles were synthesized and their potential applicationevaluated for use using photodynamic therapy as an antibacterial andcrosslinking agent.

Synthesis and Characterization of Chitosan Polymer-Rose BengalNanoparticles:

Chitosan polymer-Rose Bengal nanoparticles were synthesized byconjugating spherical chitosan nanoparticles formed using an ionicgelation method with Rose Bengal using the procedure illustrated in FIG.1 and described below. Chitosan purchased from Sigma-Aldrich, St. Louis,USA, was dissolved in 1 v/v % acetic acid solution at a concentration of0.12 w/v %, and the pH was raised to 5 with 1M NaOH. Chitosannanoparticles were formed spontaneously using an ionic gelation methodby adding 0.1% sodium tripolyphosphate in water to chitosan solution ina ratio of 3:1 under stirring at a speed of 1000 rpm for 5 minutes.

Chitosan polymer-Rose Bengal nanoparticles were thereafter synthesizedusing chemical crosslinker carbodiimides (N-ethyl-N(3-dimethylaminopropyl) carbodiimide—EDC). EDC (5 mM) 380 mg/400 mL and NHS 5 mM(230 mg/400 mL) was added followed by Rose Bengal (to get a ratio of10:1 with chitosan). The conjugation reaction was carried out in thedark. The chitosan polymer-Rose Bengal nanoparticles formed werecollected using centrifugation at 15,000 rpm for 20 minutes. Thechitosan polymer-Rose Bengal nanoparticles were then dialyzed against anacetic acid buffer (pH 5.5) using a dialysis membrane (Sigma, cellulosetubing, cut off 1200014000 g/mol). The water was changed daily anddialysis was carried out for a period of 1 week. The dialysis wasstopped when no Rose Bengal residues were detected in the UV-visiblespectrum of the dialysate.

The chitosan polymer-Rose Bengal nanoparticle filtrate was nextfreeze-dried starting at −80° C. The chitosan polymer-Rose Bengal wasformed as a dried cotton mass that was milled using a sterile glassstirrer to obtain a fine powder of nanoparticles. The nanoparticles werestored in a cool and dark place until further use.

Absorption spectra for conjugated (chitosan polymer-Rose Bengalnanoparticles) and unconjugated (Rose Bengal) solutions were recordedusing a UV-Visible spectrophotometer (Shimadzu 110e, Japan) (FIG. 8B).Photophysical characterization of chitosan polymer-Rose Bengalnanoparticles to determine the ratio of monomer to dimer (absorbance at560 nm to 528 nm) at different concentrations was also carried out. Theeffective concentration of chitosan polymer-Rose Bengal nanoparticleswas determined based on the highest monomer:dimer ratio (leastaggregation). The conjugated chitosan polymer-Rose Bengal nanoparticleswere analyzed for their chemical composition using Fourier TransformInfrared (FTIR) spectrophotometer (Shimadzu, Japan) (FIG. 8C). Theprepared chitosan polymer-Rose Bengal nanoparticles were mixed withpotassium bromide (1:100 w/w) for the FTIR spectroscopy (16 cm⁻¹resolution, 32 scans per sample).

Photo-oxidative characterization was conducted to assess the ability togenerate singlet oxygen by the chitosan polymer-Rose Bengalnanoparticles. Measurements were carried out in a 24 well plateaccording to a procedure described in Hadjur et al., J. Photochem.Photobiol., B; 45,170-178, 1998. Generation of singlet oxygen onphotoactivation of Rose Bengal and chitosan polymer-Rose Bengalnanoparticles was studied photometrically using1,3-diphenylisobenzofuran (DPBF), a singlet oxygen scavenger. 2 mL DPBF(200 μM in ethanol) was added (corresponding to absorbance intensitybetween 1.5 and 2 at 410 nm, in a 24 well plate) to 100 μL of differentphotosensitizer solutions (total volume=2.1 mL). Lumacare™ white lightsystem with 540 nm (output power=50 mW) fiber was used as a lightsource. The decrease in absorbance intensity at 410 nm was monitored asa function of time using a UVVISIBLE™ microplate reader (Epoch, Biotek,USA). The rate of singlet oxygen production was related to the rate ofdecrease of DPBF absorbance at 410 nm as a function of irradiation time(FIG. 9).

Chemically Modified Phosphorylated Micro/Nano-Chitosan (P-NC) to InduceBiomineralization:

Biomimetic mineralization is a process carried out to imitate thenatural process of mineralization, and thereby render the collage matrixof demineralized dentin remineralizable. The advantage of biomimeticmineralization is that it simulates the natural process of mineralcrystal formation on the surface of organic or inorganic matrix withoutthe need for harsh chemicals. The behavior of phosphorylatedNon-Collagenous Proteins (NCPs) in biomineralization, suggest theirsuitability in methods for biomimetic mineralization to facilitateremineralization of demineralized connective and hard tissues such asdentin.

In one embodiment, the collagen matrix of demineralized dentin is madeto work as a scaffold for remineralization. Nanoparticles ofphosphorylated chitosan P-chi of between about 40 nm to 80 nm wereprepared using commercially available chitosan (Sigma, Chemical Co. USA)with low molecular weight (75-85% deacetylated) by the reaction ofchitosan with phosphorous pentoxide, following the method developed byNishi et al. Ibusuki S, Halbesma G J, Randolph M A, Redmond R W,Kochevar I E, Gill T J. Photochemically cross-linked collagen gels asthree-dimensional scaffolds for tissue engineering. Tissue Eng. 2007August; 13(8):1995-2001. Experiments were performed on micro sizedparticles of chitosan (not nanoparticles) to test the mechanism. Themechanism of the reaction is shown in FIG. 4, where the hydroxyl groupis phosphorylated and the amino group is retained. Since thephosphorylated chitosan (P-chi) is water-soluble at pH 7.0, an aqueoussolution of P-chi was prepared for treating dentin samples.

To coat P-chi on the surface of the dentin section/dentin collagen, 50mg dentin collagen were mixed with 5 mL of P-chi solution (5 mg/mL) andthen dried in a chemical hood until the water was completely vaporized.The covalent immobilization of P-chi on the surface of the dentinsections or dentin collagen particles was carried out by putting onedentin section or 50 mg dentin collagen particles to 10 ml P-chisolution containing glutaraldehyde (GA), which is a cross-linker (Sigma,Chemical Co. USA) of 0.25% (wt %) at 4° C. for 24 h. It was found thatthe phosphorylated chitosan when treated with demineralized dentinpromoted biomineralization (FIGS. 2 to 6). The use of the compositionfor remineralizing the demineralized dentin advantageously aids not onlyreinforcement mechanically and chemically of the hard tissue, but alsoaids in promoting biomineralization of the interface subsequent tointerfacial failure and penetration of saliva.

Evaluation of Cytotoxicity of Chitosan Polymer-Rose BengalNanoparticles:

To evaluate cytotoxicity approximately 1×10⁵ NIH 3T3 mouse fibroblastcells (American Type Culture Collection CCL 1, Rockville, Md.) wereseeded into 24 well plates in Dulbecco's Modified Eagle medium (DMEM)supplemented with 10% Bovine Serum and antibiotics and incubated for 48hrs in 5% CO₂ incubator (Thermo Electron Corporation, USA). Afterincubation, chitosan polymer-Rose Bengal nanoparticles and Rose Bengaldissolved in DMEM were added into the cells and incubated for 15 min indark. The cells were irradiated with a white light source and a 540 nmfiber (Lumacare Inc) for a total dose of 20 J/cm². Rose Bengal andchitosan polymer-Rose Bengal nanoparticles were also tested withoutirradiation. The cells were left in the media for 24 hours underincubation.

The supernatant media was removed without disturbing the cell line, andthe cell layer was washed with 1 mL of phosphate-buffered saline. Cellsurvival was determined by the standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma Aldrich) thatdetermines the mitochondrial activity [34]. MTT medium was applied at aconcentration of 0.5 mg % in phosphate-buffered saline and incubated for4 hours. After the incubation period, MTT medium was removed, and 1 mLdimethyl sulfoxide was added to dissolve the insoluble formazancrystals. The absorbance at 540 nm was measured photometrically by usinga UV-visible spectrophotometer (Epoch, Biotek, USA). Percentage survivalwas calculated based on control sample without any treatment as 100%.All analyses were repeated three times in triplicate, and thestatistical significance was analyzed by one-way analysis of variance.

Uptake of Rose Bengal, MB and Chitosan Polymer-Rose Bengal Nanoparticlesby E. faecalis Biofilm:

The uptake of Rose Bengal, methylene blue and chitosan polymer-RoseBengal nanoparticles was evaluated on biofilm forms of E. faecalis. Aseven day old biofilm of E. faecalis (ATCC 29212) was grown in 24multi-well plates. 1 mL of overnight E. faecalis culture was added intoeach well of the multi-well plates and incubated at 37° C., 100 rpm.Fresh media was replenished every 48 hours to provide a constant supplyof nutrients and to remove dead bacterial cells. On the eighth day, themedia was removed from the wells, and the biofilm was carefully washedonce with sterile deionized-water. Different concentrations of chitosanpolymer-Rose Bengal nanoparticles (0.3, 0.5 & 1 mg/mL) and MB and RoseBengal (10, 25, 50 & 100 1 μM) were added to the biofilm and incubatedat 37° C. for 15 min, protected from ambient light. Three samples wereused for each concentration. Excess photosensitizer solutions wereremoved leaving behind the bound Rose Bengal, MB and chitosanpolymer-Rose Bengal nanoparticles in biofilm and washed once. Thebiofilm bacteria were treated with 1 mL of 2% SDS for 20 h in order toextract the cell-bound photosensitizers. The biofilms were disrupted andcollected in eppendorf tubes. The biofilm bacteria were centrifuged(3000 rpm, 10 min) and the supernatant solution was taken forphotosensitizer quantification. Quantification of photosensitizer wasdone spectrophotometrically (Epoch, Biotek, USA) at the absorptionmaxima of the Rose Bengal (540 nm). Calibration curves were constructedfor each Rose Bengal in 2% SDS. Uptake values were obtained as the totalRose Bengal and MB concentrations (μM) extracted from both the 1 mL ofplanktonic and biofilm bacteria.

Effect of Rose Bengal and Chitosan Polymer-Rose Bengal Nanoparticles onthe Membrane Integrity of Planktonic Bacteria:

Leakage of cytoplasmic contents (DNA) is a characteristic indication ofdamage to the bacterial cytoplasmic membrane. Absorbance at 260 nm wasused to estimate the amount of intracellular contents leaked frombacteria subjected to different photosensitizers and photodynamictherapy. E. faecalis (ATCC 29212) was incubated overnight at 37° C.under agitation in the Brain-Heart Infusion (BHI) medium (Sigma, USA).The culture was centrifuged (4000 rpm, 10 min, 4° C.), supernatantsdiscarded and washed twice in sterile deionized water (DIW). The cellswere resuspended in deionized water and adjusted to 10⁷ CFU/mL (opticaldensity 0.7) at 600 nm. The cell suspension (1 mL) was then added intoeppendorf tubes and centrifuged. The supernatants were discarded and thecell pellets were treated with different photosensitizer solutions andmaintained at 37° C. for 15 min, protected from ambient light. Thekinetics of release of intracellular contents, treated bacterial cellswere filtered (0.2 μm pore size, Pall) and absorbance of the filtraterecorded at 260 nm (OD₂₆₀). For photodynamic therapy, thephotosensitized cells were centrifuged and cell pellets irradiated (5J/cm², 540 nm). The % change in OD260 at 15 min post sensitization andafter irradiation with 5 J/cm² was calculated with respect to the OD260of the sample measured at 0 min.

Assessment of Antibacterial Efficacy of Rose Bengal and ChitosanPolymer-Rose Bengal Nanoparticles:

E. faecalis (ATCC 29212) was used to test the antibacterial efficacy ofRose Bengal and chitosan polymer-Rose Bengal nanoparticles in bothplanktonic and biofilm forms. Planktonic cell pellets (10⁹ cells/mL)were obtained and the cell pellets treated with 1 mL of Rose Bengal (10μM) and chitosan polymer-Rose Bengal nanoparticles (0.1 & 0.3 mg/mL), at37° C. for 15 min, in the manner previously described and protected fromambient light to allow sensitization. Dark toxicity was evaluated after15 min of sensitization with the two treatment solutions. In the case ofphotodynamic therapy, the photosensitizer solutions were removed leavinga thin smear at the bottom of the eppendorf tubes. The sensitizedplanktonic-bacteria were irradiated using a 540 nm fiber with doses of 2and 5 J/cm². After treatment, cell pellets were resuspended in steriledeionized-water (1 mL) and 100 μL of the suspension was plated infreshly poured BHI agar plates after serial dilution. Colonies werecounted after 24 hours of incubation at 37° C. and expressed as logcolony forming units (CFU) per mL.

In order to test the antibacterial-efficacy of nanoparticulates onbacterial-biofilm, 7-days old biofilm of E. faecalis (ATCC 29212) wasgrown in well of multiwell-plates as mentioned above. On the eighth day,the media was removed from the wells, and the biofilm was carefullywashed once with sterile deionized-water. The biofilm-bacteria wastreated with chitosan polymer-Rose Bengal nanoparticles and Rose Bengaland exposed to photodynamic therapy with different doses. Sensitizationwas done using 1 mL of Rose Bengal (10 μM) and chitosan polymer-RoseBengal nanoparticles (0.1 & 0.3 mg/mL) at 37° C. for 15 min, protectedfrom ambient light. Subsequently, the excess photosensitizer solutionswere removed leaving behind the bound chitosan polymer-Rose Bengalnanoparticles and Rose Bengal. Dark toxicity was evaluated aftersensitization period with the two treatment solutions. In case ofphotodynamic therapy, the sensitized biofilm-bacteria were irradiatedusing a 540 nm fiber with dosage of 20, 40 and 60 J/cm²; andfractionated dosage of 10 and 20 J/cm² twice. After treatment, thebiofilms were washed gently and 1 mL of sterile deionized-water wasadded. Biofilmbacteria were disrupted mechanically and plated in freshlypoured BHI agar plates following serial dilutions. Control wells weremaintained in sterile deionized-water. Colonies were counted after 24hours of incubation at 37° C. and expressed as log colony forming units(CFU) per mL. The experiments were carried out in triplicates and themean values were calculated.

Assessment of Biofilm-Structure Following Rose Bengal and ChitosanPolymer-Rose Bengal Nanoparticles Treatment:

The structure of the 7-days old biofilm following treatment withnanoparticulates was assessed using confocal-laser-scanning-microscopy(CLSM). E. faecalis (ATCC 29212) biofilm was grown on a glass bottomculture dishes. Following treatment with Rose Bengal (10 μM) andchitosan polymer-Rose Bengal nanoparticles (0.3 mg/mL) the as mentionedabove, biofilms were washed with 1 mL of sterile deionized-water. Thebiofilms were then stained with 200 μL, of Live/Dead® Baclight™ stain(Molecular Probes, Eugene, Oreg.) and incubated in the dark for 10minutes. The biofilm-structures were then viewed under spinning diskconfocal-laser-scanning microscopy (Olympus, Japan). Kr/Ar laser was thesource of illumination with 488 nm excitation and long-pass 500-523 nmand 622-722 nm emission filter settings for green and red signalsrespectively. Nine different areas were imaged from each sample using a60× oil objective. The optical sections of the biofilm-structure werefirst recorded then subsequently analyzed using Velocity® software.Student t-test was used to compare the thickness of the biofilm beforeand after nanoparticulates treatment.

Effects of Chitosan Nanoparticles and Dentin Surface Treatment withConjugated Chitosan on Biofilm Formation within the Sealer-Root DentinInterfaces:

Bacterial recolonization after treatment still remains a major concernin endodontically treated teeth. Chitosan and its variants arebiocompatible natural biopolymers, which possess versatile biologicalactivities including antibacterial properties. An assessment of biofilmformation within the sealer-dentin interfaces of roots filled withchitosan nanoparticles modified sealer and its combination with rootdentin surface treatment by phosphorylated chitosan and/orphotosensitizer conjugated chitosan (Rose Bengal-chitosan). Standardizedspecimens comprising of coronal 4 mm root segments of bovine incisors(n=17) were surface treated with the test materials and filled withgutta-percha rubber and zinc oxide eugenol (ZOE) sealer containingchitosan nanoparticles. The control group was filled with gutta-percharubber and zinc oxide eugenol sealer. After setting at 100% relativehumidity for 7 days, samples were conditioned at 37° C. for 4 weeks insimulated saliva solution. Monospecies biofilms of Enterococcus faecalis(ATCC 29212) were grown on the specimens for 7 days in a chemostat-basedbiofilm fermentor, mimicking pathogenic oral conditions. The extent ofbiofilm formation within the sealer dentin interface was assessed usingconfocal laser scanning microscopy and scanning electron microscopy.Biofilm surface area data was analyzed by Kruskal-Wallis andMann-Whitney U tests. Specimens with chitosan nanoparticles in thesealer alone (489.77±269.66 μm²) and those receiving phosphorylatedchitosan and photosensitizer conjugated chitosan/phosphorylated chitosansurface treatment (574.1±186.21 μm², 949.3±510.03 μm², respectively)showed less biofilm formation than the zinc oxide eugenol sealer controlgroup (2438.52±383.26 μm², p<0.05). Within the test model used,incorporating chitosan nanoparticles into zinc oxide eugenol sealer andthe surface treatment with phosphorylated chitosan or RoseBengal-chitosan/phosphorylated chitosan increased the resistance tobiofilm formation. The results are illustrated in FIG. 7.

Photodynamic Crosslinking of Dentin-Collagen:

Sixteen freshly extracted human incisors and eight bovine incisors werestored in 0.9% saline until use. The bovine teeth were used formechanical testing while the human teeth were used for chemical andenzymatic-degradation analysis. Dentin sections of 0.5 mm thickness wereprepared from either side of the root canal lumen using a slow speeddiamond wafering blade (Buehler, UK) under continuous water irrigation.The sections were further prepared into rectangular dimensions of12×2×0.5 mm (human) and 16×2×0.2 mm (bovine) by grinding in wet emerypaper of grit sizes 400, 800 and 1000 under continuous water irrigation.The dentin sections were demineralized in 1M EDTA (pH=7.4) for sevendays. The resulting dentin collagen specimens were rinsed for 10 minutesin deionized water to remove residual EDTA and subsequently stored insterile deionized-water at 4° C. The demineralized dentin collagenspecimens (total−48) were randomly divided into four treatment groups(n=12): 1) No-treatment—(Control); 2) 2.5% glutaraldehyde; 3) RoseBengal 10 μM; and 4) chitosan polymer-Rose Bengal nanoparticles 0.3mg/mL chitosan polymer-Rose Bengal. The dentin-collagen samples werecrosslinked with glutaraldehyde for a period of 6 hours. In photodynamiccrosslinking, collagen-samples were placed in a 24 well-plate (area of 2cm²/well) and immersed in 1 mL of Rose Bengal or chitosan polymer-RoseBengal nanoparticles solution for 15 min. After the sensitizationperiod, excess Rose Bengal and chitosan polymer-Rose Bengalnanoparticles were removed and the photosensitized collagen wasactivated with a non-coherent light (540 nm, 20 J/cm²) (LumaCare Inc.,NewPort Beach, Calif., USA). Crosslinked specimens were thoroughlywashed in deionized-water three times, stored in a vacuum dessicatorovernight and then tested for chemical analysis. For the enzymaticdegradation analysis, the specimens were lyophilized for 24 hours. Thebovine dentin-collagen specimens were maintained in deionized-water tobe used for mechanical testing.

Chemical Analysis:

The vacuum desiccated collagen specimens were treated with liquidnitrogen, ground and mixed with potassium bromide (1:100 w/w) for thefourier transform infrared (FTIR) spectroscopy (16 cm⁻¹ resolution, 100scans per sample) (Shimadzu, Kyoto, Japan).

Determination of Mechanical Properties:

Enzymatic degradation analysis was conducted to quantify the amino acidrelease using Ninhydrin assay as described by Mandl et al. Mandl, I., J.D. Maclennan, and E. L. Howes, Isolation and characterization ofproteinase and collagenase from Cl. histolyticum. J Clin Invest, 1953.32(12): p. 1323-9. The dentin-collagen specimens were subjected toenzymatic degradation using collagenase from Clostridium histolyticumwith an activity of 125 CDU/mg solid (P/N C-0130; Sigma). Desiccatedcollagen specimens (5 mg) were added into 5 mL of buffer solution (50 mMHEPES containing 0.36 mM CaCl₂) and incubated at 37° C. for 30 min., 0.1mL collagenase enzyme (0.1 mg/mL in HEPES buffer) was added into thecollagen containing buffer solution and incubated at 37° C. in anorbital incubator (100 rpm). After 1, 2, 3 and 7 days of degradation,200 μL of the solution was added into ninhydrin reagent (2 mL), mixedwell and kept in boiling water for 30 min. The containers were allowedto cool to room temperature and 10 mL of 50% isopropanol was added. Theamount of free amino acids released following degradation of collagenspecimens after heating with ninhydrin, was proportional to the opticalabsorbance (560 nm) of the solution. The amount of amino acids releasedfrom the crosslinked and non-crosslinked dentin-collagen specimens werequantified using the standard curve of L-Leucine.

Determination of Mechanical Properties:

The fully-hydrated bovine dentin collagen specimens from all four testgroups were used for tensile testing (Instron 5544™, InstronCorporation, Canton, Mass.) with a 100 N load cell. The specimens werepositioned in the loading jig by gripping the two ends (4 mm) andsubjected to tensile load at a crosshead speed of 1 mm/min until failureoccurred. Care was taken to keep the samples hydrated at all timesduring the test. The stress-strain curve per sample was plotted for allthe groups. The ultimate tensile strength and toughness (MPa),represented by the area under the stress-strain curves were calculatedusing OriginPro 8.1™ software (OriginLab Corporation, MA).

TEM Evaluation:

Four specimens from each group were processed for TEM evaluation aftercrosslinking. The collagen specimens were fixed overnight in 2.5%glutaraldehyde (0.1M phosphate buffer). All specimens for the TEM wereprepared following previous protocol [3]. The 90 nm thick sections wereprepared along the cross-section of the specimens and examined under TEM(Hitachi H-7000, Tokyo) at 80 kV.

Characterization of Polymeric Photosensitizes Chitosan Polymer-RoseBengal Nanoparticles:

FIG. 8A shows electro micrographs of the aggregates of sphericalchitosan polymer-Rose Bengal nanoparticles under TEM with 60±20 nm size.The zeta potential of the chitosan polymer-Rose Bengal nanoparticles wasfound to be +30±0.8 mV. The absorption spectra obtained for chitosanpolymer-Rose Bengal nanoparticles displayed bands characteristic of RoseBengal is illustrated in FIG. 8B. The amount of Rose Bengal uptake bythe conjugated chitosan polymer-Rose Bengal nanoparticles was calculatedto be 14 μM per 0.1 mg. This confirms that Rose Bengal is attached tothe chitosan polymer chain. FTIR spectra of conjugated chitosan RoseBengal illustrated graphically in FIG. 8C showed bands that could beassigned to the amide bonds between chitosan and Rose Bengal. Twocharacteristic peaks at 1651 (amide I, carbonyl stretching vibration)and 1558 cm⁻¹ corresponding to (NH₂ bending) were prominent in chitosanand chitosan polymer-Rose Bengal spectra (Moczek & Nowakowska, 2007).However, the ratio of intensities at 1558 and 1652 cm⁻¹ was higher inchitosan polymer-Rose Bengal when compared to chitosan suggesting thereduction of amide I bonds due to utilization of free amine groups ofchitosan to form bonds with CO— group of Rose Bengal. The peak (900-1100cm⁻¹) corresponding to the saccharide group of chitosan was alsoprominent in the chitosan polymer-Rose Bengal.

Chitosan polymer-Rose Bengal nanoparticles showed the ability to producesinglet oxygen upon photoactivation similar to Rose Bengal (FIG. 9)observed as a decrease in the DPBF concentration. The singlet oxygenrelease was high enough to convert all the available DPBF for both thephotosensitizer. The rate of singlet oxygen generation increased withincrease in the concentration of both the photosensitizer used.Following results of the characterization of chitosan polymer-RoseBengal nanoparticles, concentration of 0.3 mg/mL was used in all thesubsequent experiments.

Cytotoxicity Assay Using Rose Bengal and Chitosan Polymer-Rose BengalNanoparticles:

FIG. 10 shows the cell survival in percentage after differentphotosensitizer treatments. Chitosan polymer-Rose Bengal nanoparticlesdid not show any dark toxicity with 15 min exposure time. Followingirradiation, toxicity increased up to 50% depending upon the chitosanpolymer-Rose Bengal nanoparticles concentration used. Rose Bengal showedhigher dark toxicity and further reduction of cell survival afterphotodynamic therapy.

Uptake of Rose Bengal, Methylene Blue and Chitosan Polymer-Rose BengalNanoparticles p by E. faecalis Biofilm:

Conjugation of anionic photosensitizer (Rose Bengal) with chitosanenhanced the uptake into the bacterial cells. The Rose Bengal aloneshowed minimal diffusion in contrast to chitosan polymer-Rose Bengalnanoparticles.

TABLE 1 Uptake from bacterial cells obtained after incubation withdifferent photosensitizers. Biofilm E. faecalis photosensitizer (μM)photosensitizer uptake/mL of cells RB 10 μM 2.72 ± 0.15 RB 25 μM 2.80 ±0.09 RB 50 μM 3.01 ± 0.11 RB 100 μM 3.68 ± 0.17 MB 10 μM 0.96 ± 0.07 MB25 μM 1.75 ± 0.21 MB 50 μM 3.15 ± 0.16 MB 100 μM 5.07 ± 0.19 CSRBnp 0.3mg/mL 16.15 ± 5.82  CSRBnp 0.5 mg/mL 24.06 ± 9.77  CSRBnp 1.0 mg/mL40.68 ± 4.32 

Values represent the uptake in μM/mL of cells obtained after incubationof biofilm bacteria with Rose Bengal, methylene blue (MB) and chitosanpolymer-Rose Bengal nanoparticles. Values are the means of threereadings±standard deviations. There was a significant increase in uptakeof photosensitizer by bacterial cells when conjugated with chitosan.Biofilm showed significant increase in uptake of chitosan polymer-RoseBengal nanoparticles as compared to planktonic bacteria. (P<0.05).

The exact quantity of Rose Bengal uptake was calculated using thestandard curve of Rose Bengal in 2% SDS. Chitosan nanoparticles areknown to kill bacteria by inducing membrane permeability and subsequentleakage of intracellular components (Rabea et al. 2003). In addition,chitosan polymer-Rose Bengal nanoparticles were also found to bepositively charged and therefore more amenable to permeation. The shortexposure time to chitosan polymer-Rose Bengal nanoparticles maytherefore operate to enhance entry of the Rose Bengal into the cellsthrough the pores created by the chitosan nanoparticles.

Effect of Rose Bengal and Chitosan Polymer-Rose Bengal Nanoparticles onthe Membrane Integrity of Planktonic Bacteria:

Bacterial membrane damage and subsequent leakage of cell constituentswere higher with chitosan polymer-Rose Bengal nanoparticles than RoseBengal without photodynamic therapy as for example is illustratedgraphically in FIG. 11. Following photodynamic therapy both Rose Bengaland chitosan polymer-Rose Bengal nanoparticles showed increasedabsorbance at 260 nm Chitosan polymer-Rose Bengal nanoparticles at 0.3mg/mL showed inherently higher ability to induce bacterial membranedamage as compared to lower concentration used.

Assessment of Antibacterial Efficacy of Rose Bengal and ChitosanPolymer-Rose Bengal Nanoparticles:

FIG. 12 shows the antibacterial efficacy of Rose Bengal and chitosanpolymer-Rose Bengal nanoparticles on planktonic and biofilm-bacteria.Chitosan polymer-Rose Bengal nanoparticles showed almost completekilling of planktonic E. faecalis even after 15 min of sensitization. Intest studies, chitosan polymer-Rose Bengal nanoparticles and Rose Bengalshowed complete planktonic bacterial killing at 2 and 5 J/cm²respectively see FIG. 12, slide (A). In case of biofilm-bacteria, bothchitosan polymer-Rose Bengal nanoparticles and Rose Bengal did not showcomplete killing even at 60 J/cm² (FIG. 12, slide B). Followingfractionation of the photodynamic therapy, complete elimination ofbiofilm bacteria was obtained with chitosan polymer-Rose Bengalnanoparticles (0.3 mg/mL) and not with Rose Bengal (FIG. 12, slideC).Fractionation of light dosage during PDT enhances the availability ofmolecular oxygen by providing a lag phase to allow oxygen replenishment.Likewise the slower release of singlet oxygen as observed with CSRBnpcould provide sufficient time for molecular oxygen to be replenished inthe site of PDT and prolong the antibacterial effect. This can alsopromote deeper penetration of singlet oxygen into the biofilm structurethus, resulting in complete biofilm elimination.

Assessment of Biofilm-Structure Following Rose Bengal and ChitosanPolymer-Rose Bengal Nanoparticles Treatment:

FIG. 13 shows CLSM images (A) to (E) of the bacterial-biofilms beforeand after photodynamic therapy treatment. In the untreated control, thebiofilm-structure consisted of both live (green) and dead (red)bacterial-cells in a multilayered architecture. The number of livebacterial-cells was observed to be higher as compared to the dead cells.The thickness of biofilm-structure was found to be variable at differentlocations (39.2±7.3 μM). Both the dark toxicity and photodynamic therapytreatment groups with Rose Bengal and chitosan polymer-Rose Bengalnanoparticles showed reduction in the biofilm thickness andbiofilm-architecture was altered in case of chitosan polymer-Rose Bengalnanoparticles. Rose Bengal showed lower killing efficacy due to darktoxicity alone and irradiation resulted in higher killing of biofilmbacteria. However, the biofilm architecture was not disrupted.Distribution of viable bacteria reduced significantly and themultilayered structure as observed in the control biofilm was disruptedfollowing chitosan polymer-Rose Bengal nanoparticles treatment.Bacterial-biofilms exposed to chitosan polymer-Rose Bengal nanoparticleswere completely disrupted with conspicuous loss of the intricatethree-dimensional form after PD treatment. The thickness of the biofilmreduced significantly to 13.1±4.3 μM (p<0.01) and 21.5±9.57 μM (p<0.05)after PD treatment with chitosan polymer-Rose Bengal nanoparticles andRose Bengal respectively.

Photodynamic Crosslinking of Dentin-Collagen:

FIG. 14A shows schematically the FTIR spectra obtained fromdentin-collagen. The amide I bands (1666 cm⁻¹), amide II band (1558cm⁻¹) and CN (1458 cm⁻¹) bands are analyzed to assess the presence ofcrosslinking. Amide I bands (1666 cm⁻¹) has been attributed to C=0stretching vibrations coupled to N—H bending vibration. The amide IIbands (1566 cm⁻¹) are due to the N—H bending vibrations coupled to C—Nstretching vibrations. Following crosslinking of collagen, the amidebands specially amide I decreased as compared to the amide II bands inall the crosslinked samples. The reduced amide I peak relative to theamide II peak could be due to the conversion of the free —NH₂ groups incollagen to N—H groups. Increase in CN bands relative to amide I bandssuggests presence of crosslinking between COOH and NH₂ groups.

The amount of amino acids released following enzymatic degradation ofthe crosslinked and non-crosslinked dentin-collagen was significantlydifferent as a function of time (p<0.05) (FIG. 14B). After 7 days, thecontrol group specimens disintegrated completely and released thehighest amount of amino acid (5 μmol/mL). The GD group showed thehighest resistance to collagenase degradation even on the 7^(th) day(0.096 μmol/mL). In case of photodynamically crosslinked dentin-collagensamples using Rose Bengal, resistance to degradation was comparable tothe GD group till day 3 and showed minimal increase on day 7 (0.25μmol/mL). Chitosan polymer-Rose Bengal nanoparticles crosslinkeddentin-collagen showed slightly faster degradation as compared to RoseBengal on day 7, and which possibly associated with the degradation ofchitosan. Furthermore, the interaction of chitosan and collagen wasevaluated using SDS Page analysis (data not shown). The collagen samplestreated with chitosan showed bands similar to the collagen control evenafter exposure to collagenase enzyme. This could be due to theinteraction of collagen with chitosan resulting in covering of sitesthat are susceptible to collagenase attack. Chitosan has also been shownto neutralize matrix metalloproteinases that degrade the collagen. Thiscould provide additional protection to collagen against enzymaticdegradation.

FIG. 14C illustrates stress-strain curves demonstrating increasedultimate tensile strength and fracture toughness of all the crosslinkeddentin-collagen samples compared with the non-crosslinked controlsamples. Even though, the GD crosslinked dentin-collagen samples showedhigher increase in ultimate tensile strength the percentage elongationof the collagen-samples decreased drastically contributing to brittlebehavior. The average initial toughness of collagen followingdemineralization was 17 MP. The samples crosslinked using GD showedreduction in toughness by almost 38%. Samples from other two treatmentgroups, Rose Bengal (196%) and chitosan polymer-Rose Bengalnanoparticles (281%) showed significant increase in toughness comparedto the control group samples (p<0.05).

TEM micrographs from the control group revealed a collagen matrix thatcontained intact, banded collagen fibrils (FIGS. 15A and 15B). Followingcrosslinking using chitosan polymer-Rose Bengal nanoparticles, thearrangements of the collagen fibrils were denser with smooth edges (FIG.15C). The surface of the crosslinked collagen showed a layer of collagenfibrils and nanoparticles incorporated within the collagen mesh (FIG.15D).

Conjugation of chitosan with Rose Bengal exhibits characteristics ofboth polymer and photosensitizer as determined by the absorption andFTIR spectra of chitosan polymer-Rose Bengal. Where higher concentrationof chitosan are used, the chitosan polymer-Rose Bengal nanoparticlesconjugates are cationic in nature as a result of the free amine groups.Since higher concentrations (>0.5 mg/mL) of chitosan polymer-Rose Bengalshowed aggregation by low monomer to dimer ratio, chitosan polymer-RoseBengal nanoparticles at a concentration of about 0.01 to 0.5 mg/mL, andpreferably about 0.3 mg/mL were used. The presence of a saccharide peakin FTIR and a decreased amide I peak indicates chemical conjugation ofchitosan with Rose Bengal. The ability for chitosan polymer-Rose Bengalnanoparticles to produce singlet oxygen was seen as the reduction ofDPBF absorbance. The rate of singlet production by chitosan polymer-RoseBengal nanoparticles was less compared to Rose Bengal, and it isbelieved this may be due to the Rose Bengal bound to the polymericchitosan nanoparticles. Chitosan is a known oxygen scavenger and mayhave contributed to this reduction in the release of singlet oxygen.Rose Bengal showed higher cytotoxicity with and without photodynamictherapy, however, the chitosan polymer-Rose Bengal nanoparticles showedlesser degree of cytotoxicity even after photodynamic therapy.

Chitosan Polymer Rose Bengal Nanoparticles

Chitosan polymer nanoparticles due to their cationic charge andnano-form are highly reactive towards anionic particles or surfaces.However, the time taken to exert significant antibacterial activity iscomparatively long (48 hrs) and presents as an important limitation tobe used clinically for root canal disinfection. In addition due to itslow solubility in neutral pH, it is known to form aggregates.

PDT using different photosensitizers also possesses limitations inachieving complete disinfection of root canals. This has beencontributed to the inability of PS to penetrate into the biofilmstructure, self-quenching when PS is used in high concentration andremaining PS may be toxic to the host cells. The presence of serumproteins is known to reduce the antibacterial effect of PDT. Previously,it has been proposed that by modifying chitosan with Rose Bengal (RB),the particles obtained were water soluble as well as retained the PSproperties.

Chitosan polymer Rose Bengal nanoparticles synthesised in accordancewith the present study achieved enhanced uptake into the biofilmstructure. Subsequent photoactivation of the Rose Bengal resulted in theproduction of singlet oxygen. The synergistic activity of chitosannanoparticles and photosensitizers (Rose Bengal) covalently conjugatedto it resulted in significant antibacterial activity as well asdisruption of the biofilm structure.

In addition, as shown in FIG. 16, the effect of tissue inhibitors, suchas pulp, proteins/exudates and dentine collagen matrix inhibited theantibacterial properties of chitosan nanoparticles and PDT alone. It ishighly pertinent to realize the importance of dentin constituents;tissue remnants and serum products present within the root canalsneutralize the commonly used antibacterial disinfectants. Similarreductions in the antibacterial activities of newer disinfection agentswere also reported. Till date there is no such antibacterial agentavailable for root canal disinfection that is effective even in thepresence of various tissue inhibitors. The chitosan conjugated RoseBengal nanoparticles show the ability to overcome the inhibitionfollowing photoactivation and prolonged interaction time. This furthersupports the immediate antibacterial of PDT and the delayedantibacterial effect provided by the chitosan nanoparticles.

CONCLUSIONS

The experimental results suggest that conjugation of photosensitizerswith positively charged molecules allows cationic chitosan polymer-RoseBengal nanoparticles bound actively to negatively charged bacterialsurfaces, thereby allowing enhanced penetration of the Rose Bengalthrough bacterial membranes. Without being bound by a particular theory,the close proximity is believed to advantageously facilitate thediffusion of singlet oxygen into the cells. In particular, singletoxygen is known to diffuse approximately 50 nm, with the result thatsuch close proximity would advantageously achieve more effectivebacterial elimination. In addition, chitosan in itself possesses asignificant broad-spectrum antimicrobial activity against bacteria,yeasts as well as viruses. Membrane damage, increased permeability andintracellular leakage are the antibacterial mechanisms of chitosan. Thisappears evident by the cytoplasmic release following bacterial membranedamage upon chitosan polymer-Rose Bengal nanoparticles treatment. Athigher concentrations of chitosan polymer-Rose Bengal nanoparticles, theamount of cytoplasmic release is similar to that of a photodynamictherapy, suggesting the membrane damage effect of chitosannanoparticles. Even though complete elimination of planktonic bacteriawas observed with chitosan polymer-Rose Bengal nanoparticles treatmentalone, biofilm bacteria showed a higher degree of resistance. Studieshave shown that Rose Bengal may not completely eliminate biofilmbacteria as compared to the cationic photosensitizer methylene blue.Chitosan polymer-Rose Bengal nanoparticles combined with photodynamictherapy showed complete elimination of the biofilm, which may occur as aresult of the better association of photosensitizer with the bacterialcells. The slower release of singlet oxygen by chitosan polymer-RoseBengal nanoparticles appears to aid in the elimination of biofilm duringfractionation of dosage.

The experimental results show that crosslinking delays the enzymaticdegradation of dentin-collagen, and at the same time increases theoverall UTS and fracture toughness. The chemical composition andpresence of collagen crosslinking were confirmed using FTIRspectroscopy. The tensile testing used provided information on themechanical properties such as UTS and toughness afterchemical/photodynamic crosslinking of dentin-collagen specimens. Again,without being bound to a particular theory, the shift in peak maxima ofamide I and amide II, after collagen crosslinking, has been linked tothe conversion of free —NH₂ groups to NH groups. The increase in CNbands relative to amide I bands has been suggested as due to thecrosslinking between COOH and NH₂ groups. The overlap of the amide bandsof collagen and chitosan (1589 cm⁻¹) further may result in the shift inamide II following crosslinking of dentin-collagen with chitosanpolymer-Rose Bengal nanoparticles. Bacterial collagenase enzymes degradecollagen by hydrolyzing the peptide bond on the amino-terminal side ofGlycine (—X-Gly-Pro). Commercially available purified bacterialcollagenase has been used previously to degrade collagenous tissues.Following crosslinking of collagen, the sites of collagenase attack maybe hidden or modified, and this contributes to the significantdifference in the release of amino acid residues following enzymaticdegradation. In experimental studies, untreated control specimens showedthe highest overall release of amino acid at all time points in thedegradation analysis. The ultrastructure of dentin collagen also showedincorporation of chitosan polymer-Rose Bengal nanoparticles into thecollagen matrix following crosslinking. Apart from improving theresistance of collagen, chitosan may play a role in neutralizing MMPs,which are known to degrade dentin-collagen.

Crosslinked collagen-specimens demonstrated improved mechanicalproperties with the chitosan polymer-Rose Bengal nanoparticles groupshowing the highest value of toughness, when compared to the merelycrosslinked collagen specimens. It was found that infiltration ofchitosan reinforced the collagen structure by amplifying the number ofamine reaction sites resulting in the formation of ionic complexesbetween chitosan and collagen during crosslinking. Chitosan polymershave been considered structurally similar to extracellular matrixmaterials, showing controlled cell growth and reinforcing the collagenconstructs. Incorporated chitosan may also serve as spacer blocks forsome amine groups in collagen and prevent undesired zero-lengthcrosslinking, to subsequently improve the fracture toughness.

Chitosan based micro and nanoparticles conjugated with photosensitizermolecules in accordance with the present invention provide variousadvantages in dental therapies and preferably as part of an endodontictreatment process. When activated with light (photodynamic therapy) thenanoparticles achieve the dual functions of effectively eliminatingbacterial biofilms, and further result in crosslinking thedentin-collagen. The crosslinking of collagen induced by thephotosensitizer molecule conjugated to the chitosan, allows forsimultaneous incorporation of chitosan nanoparticles into the dentinhard tissue matrix. This advantageously improves the mechanical andchemical stability of dentin.

The experiment results also highlight the ability of phosphate groupbound nanoparticles to induce biomineralization, in combination withantimicrobial properties which inhibit microbial biofilm formationwithin the interfaces. The nanoparticles of the present invention mayadvantageously be used in vivo as a step treatment strategy to treat avariety of types of infected hard tissues in clinical scenarios, whereintissue disinfection and/or structural integrity needs to be addressed.

Although the detailed description describes the use of Rose Bengal as aphotosensitizer, the invention is not so limited. It is to beappreciated that other compounds which operate to generate singletoxygen as part of photodynamic therapies may also be used. A range ofphotosensitizers, either cationic or anionic could be conjugated orencapsulated using chitosan nanoparticles. Such compounds may includewithout restriction, flavins, methylene blue, porphyrins as well asother photosensitizers that contain free reactive group in its chemicalstructure. The conjugated micro/nanoparticles could target bacteria ormammalian cells depending on the application.

Although the detailed description describes the use of the nanoparticlesin pre-treating dentin as part of an endodontic procedure, the inventionis not so limited. The particles of the present invention could equallybe used in a variety of other dental restorative applications,including, without restriction, the placement of dental filings, incrown and veneer procedures, as well as in the pre-treatment ofconnective tissues, for example, prior to the placement of dentalimplants in a patient's jaw bone.

Similarly, while the detailed description describes the use ofnanoparticles as a pre-treatment material for dentin tissue, it isunderstood that the particles may also be used with a variety of othertypes of hard and connective tissues in the body, including managementof dentin caries.

Although the detailed description describes and illustrates variouspreferred embodiments, the invention is not limited to the preferredembodiments which are disclosed. Many modifications and variations willoccur to persons skilled in the art. For a definition of the invention,reference may be had to the appended claims.

What is claimed is:
 1. A composition for endodontic or dentalrestorative use, comprising: multifunctional biopolymer-based particleswherein a first portion of biopolymer-based particles have biopolymerrepeat units covalently functionalized with photosensitizer moieties anda second portion of the of biopolymer-based particles have biopolymerrepeat units covalently functionalized with phosphorylated moieties; anda pharmaceutically acceptable liquid carrier, wherein themultifunctional biopolymer-based particles are mixed with thepharmaceutically acceptable liquid carrier to form a slurry.
 2. Thecomposition of claim 1 wherein the first portion of biopolymer-basedparticles correspond to the second portion of biopolymer-basedparticles, such that the biopolymer-based particles are functionalizedwith both the photosensitizer moieties and the phosphorylated moieties.3. The composition of claim 1 wherein the biopolymer is any one ofchitosan and chitosan-based derivatives.
 4. The composition of claim 1wherein the photosensitizer moieties are any one of an anionicphotosensitizer, a cationic photosensitizer, a porphyrin, andphotosensitizers that contain a free reactive group in its chemicalstructure, and wherein the photosensitizer moieties are selected toproduce singlet oxygen upon photodynamic therapy.
 5. The composition ofclaim 2 wherein the photosensitizer moieties are anionicphotosensitizers.
 6. The composition of claim 5 wherein the anionicphotosensitizer is any one of Rose Bengal and Flavin.
 7. The compositionof claim 1 wherein the multifunctional biopolymer-based particles have adiameter from about 40 nm to about 150 nm.
 8. The composition of claim 1wherein the multifunctional biopolymer-based particles are in an amountof about 0.3 to 1% (by weight).
 9. The composition of claim 1 whereinthe pharmaceutically acceptable liquid carrier is any one of water andan alcohol.
 10. The use of the composition of claim 1 as an antibiofilmagent for elimination and/or prevention of bacterial biofilm.
 11. Theuse of the composition of claim 1 as an endodontic agent forpre-treatment of dentin tissues.
 12. The use of the composition of claim1 in management of dentin caries.
 13. The use of the composition ofclaim 1 as a remineralization agent.
 14. The composition of claim 6wherein the multifunctional biopolymer-based particles have a diameterbetween about 40 nm to 90 nm.
 15. The use of the composition of claim 1as a stabilization agent for stabilizing or improving dentin mechanicaland chemical properties.
 16. A method of dental treatment, comprising:contacting a dentin of a tooth with a pharmaceutical compositioncomprising a slurry of biopolymer-based particles wherein a firstportion of biopolymer-based particles have biopolymer repeat unitscovalently functionalized with photosensitizer moieties and a secondportion of the of biopolymer-based particles have biopolymer repeatunits covalently functionalized with phosphorylated moieties; andexposing the slurry of biopolymer-based particles to a light having awavelength selected to activate the photosensitizer moieties for aperiod of time sufficient to crosslink the biopolymer-based particlesfunctionalized with the photosensitizer moieties to the dentin.
 17. Themethod of claim 16 wherein the first portion of biopolymer-basedparticles correspond to the second portion of biopolymer-basedparticles, such that the biopolymer-based particles are functionalizedwith both the photosensitizer moieties and the phosphorylated moieties.18. A method of dental treatment, comprising: removing infected pulptissue from a tooth root and forming a hollowed-out root canal therebyexposing dentin along a substantial length of the hollowed-out rootcanal; applying a slurry of biopolymer-based particles to the exposeddentin within the hollowed-out root canal, wherein a first portion ofbiopolymer-based particles have biopolymer repeat units covalentlyfunctionalized with photosensitizer moieties and a second portion ofbiopolymer-based particles have biopolymer repeat units covalentlyfunctionalized with phosphorylated moieties; exposing the slurry ofbiopolymer-based particles to a light having a wavelength selected toactivate the photosensitizer moieties for a period of time; and afterexposing the slurry to the light, filling the hollowed-out root canalwith a filling material.
 19. The method of claim 18 wherein the firstportion of biopolymer-based particles correspond to the second portionof biopolymer-based particles, such that the biopolymer-based particlesare functionalized with both the photosensitizer moieties and thephosphorylated moieties.
 20. The method of claim 18 further comprisingthe step of cleaning and shaping the hollowed-out root canal.
 21. Themethod of claim 18 wherein the period of time of exposure to the lightis from about 2 minutes to about 12 minutes.
 22. The method of claim 18further comprising the step of washing the hollowed-out root canalfollowing photoactivation of the photosensitizer moieties.